Counter-current chromatography (CCC) is gaining popularity as a viable separation technique, particularly in natural products chemistry. For example, high-speed counter-current chromatography (HSCCC) and centrifugal partition chromatography (CPC) have increasingly been used to isolate and purify a multitude of natural products [1-14]. Despite its indisputable worth, CCC has often been passed over for other chromatographic techniques. The lack of a clear method for Characterizing the properties and comparing the relative merits of the many possible biphasic solvent systems that might be used for CCC appears to be a major drawback to the use of CCC separations. The choice of solvent system for CCC separations is of utmost importance to the successful use of the method. Compared to the far more popular solid-support chromatography, the selection of CCC solvent systems is significantly more challenging because it is equivalent to simultaneously choosing both the column and the eluant.
Many CCC solvent systems have been proposed, studied and successfully employed over the years as reviewed in several articles [15-19]. Even though CCC is a high-resolution chromatographic method, it will not separate desired target analytes in any appreciable way unless the solvent system has been chosen very carefully. There is a window of opportunity present in CCC separations that is related to the KD value of a given chemical species, typically a molecule, in a particular solvent system. The distribution constant, KD, for a given chemical species can be expressed as the concentration of that chemical species in the stationary phase divided by the concentration of the chemical species in the mobile phase. A solvent system, where the KD value of a particular chemical species is close to one, is generally considered to be the ideal system for separating that particular chemical species with optimal resolution. From this perspective, the varied chemical constituents of a particular mixture can be schematically arrayed along a polarity continuum as a function of KD of the chemical constituents in a given solvent system. In such an array for a given solvent system, CCC separation targets an interval of the polarity continuum, called the “sweet spot” of optimal resolution [20], and the solvent system can be used in CCC to separate the chemical constituents that fall within this sweet spot with high resolution. In order to represent the position of the sweet spot in a polarity continuum, two different schematics have been developed.
Use of a CCC technique called elution-extrusion (EECCC) [21-24] has the effect of maximizing the width of the sweet spot for a given solvent system and, at the same time, minimizing run times. Under elution-extrusion conditions, compounds already separated in the column are eluted (ideally) without further change in resolution. The KD value of each chemical species can be calculated from its retention time and the appropriate parameters, as is known in the art. EECCC chromatograms of complex mixtures wherein the mixture consistutents exhibit a with a wide range of polarities tend to have a cluster of chemical species eluted near the void volume (0<KD<0.25) and another cluster near the end of the run where the last component(s) are eluted (16<KD<∞ in this example). In between the two extremes lies the sweet spot (0.25≦KD≦16) where optimal resolution of compounds is observed.
Biphasic solvent systems for CCC applications have traditionally been organized as solvent families that are comprised of the same solvents mixed in varying proportions. Common solvent families are hexane/ethyl acetate/methanol/water (HEMWat), chloroform/methanol/water (ChMWat), and heptane/ethyl acetate/methanol/water (the “Arizona” family) [25]. Solvent system families provide a methodical means of searching for a particular solvent system that predicts a reasonable KD value for the target compound(s) in a CCC separation. For example, if a relatively high concentration of a particular chemical species is determined to be present in the upper phase of the HEMWat system 0 (see Table 1, where system 0 contains equal relative proportions of the four solvents of the system family), the KD value of that chemical species will likely be brought closer to 1 (an equal concentration of the compound in both phases) by decreasing the ratios of ethyl acetate to hexane and/or water to methanol as in a system such as HEMWat−3. In this way, once a particular solvent system family member has been tested as portal to the solvent system family, there exists a methodical way to modifying the solvent system to seek one that will exhibit an optimal KD value for the target chemical species.
A factor that distinguishes one solvent system family from another is the polarity range of chemical species for which the solvent system family may be optimized. For example, the HEMWat solvent system family is generally considered to separate compounds of lipophilic to moderate polarity, while the ethyl acetate/n-butanol/water is a solvent system family that is likely to separate compounds of moderate to hydrophilic polarity. As the aforementioned example suggests, there may be considerable overlap of polarity ranges between solvent system families that introduces a degree of empiricism to the solvent system selection process.
It is important to note that in liquid/liquid chromatography separation is driven by the relative solubilities of the analytes in the two different solvent layers, and not strictly by their relative polarities. In fact, CCC has been shown to be an excellent technique for separating homologues with very similar polarities [26, 27]. However, the concept of relative polarity does provide a convenient framework with which to represent the separation potential of organic compounds.
Hitherto, no standard method existed to evaluate solvent system or solvent system family performance and, therefore, the solvent system selection process has been essentially empirical in nature.
The present invention provides a systematic way to evaluate solvent system performance and make rational selections of one or more solvent systems for the separation of constituents (unknown or known) in a given mixture.